Publications by authors named "Jingya Di"

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Efficient mutagenesis targeting the gene in mice using a combination of Cas9 protein and dual gRNAs.

Am J Transl Res 2021 15;13(10):12094-12106. Epub 2021 Oct 15.

Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University Nanjing 210046, Jiangsu, China.

We injected mouse zygotes with combinations of Cas9 protein, mRNA, and two gRNAs targeting a single exon of type I interferon receptor () to determine the gene targeting efficiencies. Cas9 protein produced on-target mutations more efficiently than mRNA when each was used with a single gRNA, regardless of which gRNA was used. When mRNA and Cas9 protein were co-injected, the on-target efficiency could reach 97.0% when both gRNAs were used, which was higher than when either gRNA was used alone (61.3% and 75.5%, respectively; P<0.05). Co-injection of Cas9 protein with both gRNAs produced the highest on-target mutation rate of any combination (100.0%). Most on-target mutations were deletions of 2 to 113 nucleotides, and there were few off-target mutations in mutant animals. The expression intensity of IFNAR1 was reduced in heterozygous mice (IF) and almost or completely absent in homozygous null mice compared with that in wild-type mice (IF and Western blot). When both gRNAs targeting were used simultaneously with two gRNAs targeting , the on-target editing efficiency on each gene was 96.8% and 85.5%, respectively. Co-injection of dual gRNAs and Cas9 protein is an efficient approach for knockout and multi-gene editing in mice and may be applied in other animal models and breeding livestock.
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October 2021